This invention relates to immunoassays and the use of nucleic acid amplification as a reporting means of the detection of an anlyte. More specifically analyte detection is achieved by forming an analyte dependent reporter-complex (ADRC), joining the reporter labels of the (ADRC), amplifying the joining product of the two labels and detecting the amplification product. Nucleic acid-labels are used to report analyte specific binding. Amplification is achieved by replication of the joined nucleic acid-labels. The method reduces the background signal of a binding assay, thereby, providing means of a highly sensitive analyte detection.
The development of immunoassays and advances in methods of nucleic acid amplification have significantly advanced the art of the detection of biological analytes. In spite of these advances, nonspecific binding of the analyte to be detected and general assay noise has remained a problem that has limited the application and sensitivity of such assays. Methods for the reduction of background noise are continually being sought.
The introduction of immunoassays in the 1960""s and 1970""s greatly increased the number of analytes amenable to precise and accurate measurement. Radio-immunoassays (RIAs) and immunoradiometric (IRMA) assays utilize radioisotopic labeling of either an antibody or a competing analyte to measure an analyte. Detection systems based on enzymes or fluorescent labels were then developed as an alternative to isotopic detection systems. D. L. Bates, Trends in Biotechnology, 5(7), 204 (1987), describes one such method based upon enzyme amplification. In this method a secondary enzyme system is coupled to a primary enzyme label. For example, the primary enzyme can be linked catalytically to an additional system such as a substrate cycle or an enzyme cascade. Enzyme amplification results from the coupling of catalytic processes, either by direct modification or by interaction with the product of the controlling enzyme.
U.S. Pat. No. 4,668,621 describes utilization of an enzyme-linked coagulation assay (ELCA) in an amplified immunoassay using a clotting cascade to enhance sensitivity. The process involves clot formation due to thrombin activated fibrin formation from soluble fibrinogen and labeled solubilized fibrinogen. Amplification of the amount of reportable ligand attached to solid-phase is obtained only by combining use of clotting factor conjugates with subsequent coagulation cascade reactions.
Substrate/cofactor cycling is another variation of enzyme-mediated amplification, and is based on the cycling of a cofactor or substrate that is generated by a primary enzyme label. The product of the primary enzyme is a catalytic activator of an amplifier cycle that responds in proportion to the concentration of substrate and hence the concentration of the enzyme label. An example of this type of substrate cycling system is described in U.S. Pat. No. 4,745,054.
Vary et al., Clin. Chem., 32, 1696 (1986) describes an enzyme amplification method suited to nucleic acid detection. This method is a strand displacement assay which uses the unique ability of a polynucleotide to act as a substrate label which can be released by a phosphorylase.
Bobrow et al., J. of Immunol. Methods, 125, 279 (1989) discloses a method to improve detection or quantitation of an analyte by catalyzed reporter deposition. Amplification of the detector signal is achieved by activating a conjugate consisting of a detectably labeled substrate specific for the enzyme system, wherein said conjugate then reacts with the analyte-dependent enzyme activation system to form an activated conjugate which deposits wherever receptor for the conjugate is immobilized.
Nucleotide hybridization assays have been developed as a means for detection of specific nucleic acid sequences. U.S. Pat. No. 4,882,269 discloses an amplified nucleic acid hybridization assay in which a target nucleic acid is contacted with a complementary primary probe having a polymeric tail. A plurality of second signal-generating probes capable of binding to the polymeric tail are added to achieve amplified detection of the target nucleic acid. Variations of this methodology are disclosed in PCT Application WO 89/03891 and European Patent Application 204510, which describe hybridization assays in which amplifier or multimer oligonucleotides are hybridized to a single-stranded nucleic acid unit which has been bound to the targeted nucleic acid segment. Signal amplification is accomplished by hybridizing signal-emitting nucleic acid bases to these amplifier and multimer strands. In all of these disclosures amplification is achieved by mechanisms which immobilize additional sites for attachment of signal-emitting probes.
Journal of Clinical Microbiol. 28, 1968 (1990) describes a system for detection of amplified Chlamydia trachomatis DNA from cervical specimens by fluorometric quantitation in an enzyme immunoassay format which includes a polymerase chain reaction.
U.S. Pat. No. 5,665,539 describes a novel system and method for sensitive analyte detection using immuno-PCR. This consists of a biotinylated DNA which binds to analyte-dependent reporter-complex via a protein A-streptavidin chimeric protein. A segment of the DNA label is amplified by polymerase chain reaction and the products are detected by agarose gel electrophoresis.
In WO 9315229, Applicants disclose a method for the detection of an analyte through the formation of a complex comprising an analyte bound to a reporter having a nucleic acid label attached. Detection of the analyte is effected through amplification of the nucleic acid label.
It is the objective of the art to increase the sensitivity of analyte detection through the use of various novel signal generating reporter conjugates and amplification strategies. However, non-specific binding-signal due to non-selective binding of reporter conjugates to walls of the reaction tubes or to solid-phase reagents used in the assays even in the absence of analyte, is a serious problem in immunoassays. Non-specific binding signal thus diminishes the ratio of the analyte specific binding to analyte non-specific binding. This reduces the sensitivity of the detection limit for an analyte. The art has identified many factors that contribute to non-specific binding such as, protein-protein interaction, adsorptive surface of the solid-phase, Vogt et al., J. of Immunological Methods, 101, 43 (1987), the assay milieu and the efficiency of the wash solution.
To resolve this problem a number of approaches have been used in this art by Vogt et al., J. of Immunological Methods, 101, 43 (1987), Graves, J. of Immunological Methods, 111, 167, (1988), Wedege et al., J. of Immunological Methods, 88, 233, (1986), Bodmer et al., J. of Immunoassay, 11, 139, (1990), Pruslin et al., J. of Immunological Methods, 137, 27, (1991), Balde et al., J. of Biochem. and Biophys. Methods, 12, 271, (1986), Hauri et al., Analytical Biochemistry, 159, 386 (1986), Rodda et al., Immunological Investigations, 23, 421, (1994), Tovey et al., Electrophoresis, 10, 243, (1989), Kenney et al., Israel Journal Of Medical Sciences, 23, 732, (1987), Hashida et al., Analytical Letters, 18, 1143, (1985), Ruan et al., Ann Clin Biochem, 23, 54, (1985). To saturate the adsorptive surface, these investigators have used blocking agents such as, proteins bovine serum albumin (BSA), gelatin, casein, non-fat dry milk, polymers (poly vinyl alcohol) detergents (Tween 20), modified antibodies (Fabxe2x80x2 and F(abxe2x80x2)2), and combinations of blocking agents (BSA, Tween 20) and pentane sulfonate. These proteins have been chosen largely by convenience and empirical testing in ELISA systems, Vogt et al., J. of Immunological Methods, 101, 43 (1987).
Despite the numerous attempts in this art to use these approaches either individually or in combination, non-specific binding has not been eliminated. Therefore, increased assay detection sensitivity has been limited. Thus, there is a continuing, unmet need for a means to reduce assay background response and to improve the signal to noise ratio of binding assays. The Applicant""s invention is the first to apply assay principle that essentially distinguishes between analyte-specific binding reagent recognition and non-specific reagent binding.
The present invention provides a method for the detection of a specific analyte. In its most basic form the method involves the formation of analyte dependent reporter complex that comprises (i) an analyte and (ii) at least two reporter conjugates bound to the analyte. Each reporter conjugate further comprises a member of a binding pair (such as an antibody) and a nucleic acid fragment or label which is joined to the member of the binding pair. The binding of the both of the reporter conjugates to the analyte operates to bring the nucleic acid labels in close proximity to each other where they may be amplified by means well known in the art. The generation of amplification products is an indication the presence of the analyte. The effect of the instant method is to provide a means of improving the ratio of the analyte-specific signal to analyte non-specific background signal, thus enhancing the ability to detect analytes at very low concentrations.
The invention may be carried out in a variety of formats including a heterogeneous format, a homogeneous format and a multianalyte binding assay format.
The heterogeneous assay format comprises the steps of: a) forming an analyte dependent reporter-complex by equilibrating an analyte to be detected, either simultaneously or in sequence, with a solid-phase analyte capture reagent and at least two reporter conjugates labeled with different nucleic acids; b) forming an analyte specific amplicon (ASA) by selectively joining the nucleic acid-labels of the reporter conjugates bound to the analyte and replicating the ASA); c) forming a nucleic acid product by amplification of the analyte-specific amplicon; d) and detecting the amplified nucleic acid products by sequence and/or size.
The homogeneous assay format will comprise the steps of: a) forming an analyte dependent reporter-complex by equilibrating an analyte to be detected, either simultaneously or in sequence, with at least two reporter conjugates labeled with different nucleic acids; b) forming an analyte-specific amplicon by selectively joining the nucleic acid-labels of the reporter conjugates bound to the analyte and replicating the ASA; c) forming a nucleic acid product by amplification of the analyte specific amplicon; d) and detecting the amplified nucleic acid products by sequence or/and size.
Finally the multianalyte assay format will comprise the steps of: a) forming simultaneously multiple analyte-dependent reporter-complexes by equilibrating analytes to be detected, either simultaneously or in sequence, with solid-phase analyte capture reagent(s) and one set of reporter conjugates for each analyte to be detected; b) forming analyte-specific amplicons by selectively joining the nucleic acid-labels of the reporter conjugates selectively bound to each analyte and replicating the ASA; c) forming nucleic acid products by selective amplification of the analyte-specific amplicons; d) and detecting the amplified nucleic acid products by sequence and/or size.
In conjunction with the above recited formats the present method may effect the amplification of the analyte-specific amplicons in several ways. For example, the amplicon may be formed by the overlap of the nucleic acid labels, or they my be joined enzymatically by means of ligation. Finally, the amplicon may be formed by either method and detected directly, with out amplification by means of signal generating substance.
Thus in one embodiment incorporating ASA formation by nucleic acid overlap, the present invention provides a method for the detection of an analyte comprising: (i) contacting an analyte having at least two reporter conjugate binding sites with at least two reporter conjugates, said reporter conjugates each comprising: a) one member of a binding pair having specificity for at least one reporter conjugate binding site on said analyte; b) a nucleic acid label; wherein said analyte binds to said reporter conjugate forming an analyte dependent reporter complex and wherein said nucleic acid labels on said reporter conjugates are joined by an overlap at each 3xe2x80x2 end, forming an analyte specific amplicon; (ii) contacting said analyte specific amplicon with a replication composition wherein said amplicon is amplified forming amplification products; and (iii) detecting said amplification products. Additionally the invention provides that the analyte may be optionally immobilzed prior to complexing with the reporter conjugate(s).
Similarly in an alternate embodiment incorporating amplicon formation by nucleic acid ligation, the present invention provides a method for the detection of an analyte comprising: (i) contacting an analyte having at least two reporter conjugate binding sites with at least one reporter conjugate pair, said reporter conjugate pair comprising a first reporter conjugate and a second reporter conjugate, each of said first and second reported conjugates further comprising: a) one member of a binding pair having an affinity for at least one reporter conjugate biding site on said analyte; b) a nucleic acid label wherein said analyte binds to said reporter conjugate forming an analyte dependent reporter complex and wherein said nucleic acid label of said first reporter conjugate comprises a 3xe2x80x2 hydroxyl group and wherein said nucleic acid label of said second reporter conjugate comprises a 5xe2x80x2 phosphoryl group and wherein said first and second nucleic acid labels are enzymatically joined to form an analyte specific amplicon; (ii) contacting said analyte specific amplicon with a replication composition wherein said amplicon is amplified forming amplification products; and (iii) detecting said amplification products. This embodiment also provides that the analyte may be optionally immobilzed prior to complexing with the reporter conjugate(s).
In another embodiment the amplicon may be detected directly by a method for the detection of an analyte comprising: (i) contacting an analyte having at least two reporter conjugate binding sites with at least two reporter conjugates, said reporter conjugates each comprising: a) one member of a binding pair having specificity for at least one reporter conjugate binding site on said analyte; b) a nucleic acid label; wherein said analyte binds to said reporter conjugate forming an analyte dependent reporter complex and wherein said nucleic acid labels on said reporter conjugates are joined, forming an analyte specific amplicon; (ii) contacting said analyte specific amplicon with a replication composition comprising a nucleic acid reporting agent, wherein said amplicon is extended and said nucleic acid reporting agent is incorporated into said amplicon; and (iii) detecting said amplicon.
A number of aspects of the present invention are unique as compared to the art. First, two nucleic acid sequences are used as reporter labels. For detection of the analyte, these must be joined together. In this way, joining forms an analyte-specific amplicon whose sequence is unique from that of reporter conjugate labels. The ASA thus functions as a surrogate for the analyte to be detected. Second, three binding-pair reagents are used in contrast to two used by other assays. One binding-pair reagent is for capturing the analyte and two are used for reporter conjugates. Third, the amplification by PCR is used to detect the product of two joined nucleic acid-label in contrast to the amplification product of a single reporter label. Fourth, the random distribution of the non-specifically bound antibodies makes them less likely to be in close proximity to an analyte binding site to interfere with interaction and joining of nucleic acid-labels. Fifth, background is minimized, or is potentially eliminated, improving sensitivity.
The specific binding enables the DNA labels to be in close proximity to each other for coupling of the nucleic acid-labels. The labels can be DNA, RNA, or DNA-RNA hybrid. The PCR amplification product can be detected by various methods known in the art, such as, gel electrophoresis, hybridization of sequence-specific probes or detected indirectly through incorporation of fluorophores, ligands or haptens.